HU222199B1 - Peptid-aldehyde derivatives of anticoagulant activity and pharmaceutical compositions containing them - Google Patents

Abstract

The present invention relates to novel peptide aldehyde derivatives of formula (I) - D-Xaa-Pro-Arg-H (I) wherein Xaa is 2-cycloheptyl-2-hydroxyacetyl or 2-cyclopentyl-2-hydroxyacetyl. , Pro is an L-proline radical and Arg is an L-arginine radical - and their acid addition salts with organic or inorganic acids, and pharmaceutical compositions containing the above compounds. The compounds of general formula (I) according to the invention have an inhibitory effect on coagulation and platelet function and on the formation of thrombosis. ŕ

Description

The present invention relates to novel peptide aldehyde derivatives of formula (I): D-Xaa-Pro-Arg-H (I) wherein

Xaa is 2-cycloheptyl-2-hydroxyacetyl or 2-cyclopentyl-2-hydroxyacetyl,

Pro is an L-proline residue and

Arg is the L-arginine residue and its acid addition salts with an organic or inorganic acid, and pharmaceutical compositions containing the above compounds.

The compounds of formula (I) of the present invention have valuable therapeutic properties, in particular anticoagulant and platelet function inhibition and thrombosis formation.

Particularly valuable examples of the compounds of formula (I) of the present invention include:

D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-L-argininaldehyde hydrogen sulfate

D-2-Cyclopentyl-2-hydroxyacetyl-L-prolyl-L-argininaldehyde Hydrogen Sulphate Definitions

The abbreviations for hydroxy and amino acids, as well as substituents and peptides formed therefrom, as used herein, correspond to those commonly used in the peptide chemical literature.

Amino acids: Arg = L-arginine [(2R) -2-amino-5-guanidino-pentanoic acid], Asp = L-aspartic acid [(2S) -2-amino-3-carboxypropionic acid], boroArg = L-boro- arginine [(R) -1-amino-4-guanidino-butylboronic acid], D-Chg = D2-cyclohexylglycine [(2R) -2-aminocyclohexylacetic acid], D-cHga = D-2- cycloheptyl-2-hydroxy-acetic acid [(2R) -2-cycloheptyl-2-hydroxy-acetic acid], D-cPga = D-2-cyclopentyl-2-hydroxy-acetic acid [(2R) -2-cyclopentyl-2-hydroxy-acetic acid ], Gla = gamma-carboxy-L-glutamic acid [(2S) -2-amino-4,4-dicarboxy-butyric acid], Glu = L-glutamic acid [(2S) -2-amino-4-carboxyl-butyric acid], Gly = glycine (2-aminoacetic acid), D-Hma = hexahydro-D-mandelic acid [(2R) -2-cyclohexyl-2-hydroxyacetic acid], D-MePhe = N-methyl-3-phenylD-alanine [(2R) ) -2-methylamino-3-phenylpropionic acid], DMePhg = DN-methylphenylglycine [(2R) -2-amino-2-phenylacetic acid], Nal (1) = 3- (naphthyl-1) -L-alanine [(2S) -2-amino-3- (naphthyl-1) -propionic acid, D-Phe = 3-phenyl-D-alanine [(2R) -2-amino-3-phenyl-propionic acid], Pro = L-proline [(2S) -pyrrolidine-2-carboxylic acid],

Substituents: Ac = acetyl, Boc = tert-butoxycarbonyl, Bz = benzoyl, Bzl = benzyl, Me = methyl, 4MeP = 4-methylpentanoyl, pNA = p-nitrophenylamino, THP = tetrahydropyranyl, Tos = p-toluenesulfonyl, Z = benzyloxycarbonyl.

Peptides and Derivatives: Abbreviations of amino acids per se denote L-amino acid. The D-amino acid is specifically designated, for example, 3-phenyl-D-alanine = D-Phe. The dash before and after the amino acid abbreviations indicates that the amino group has one H atom and the carboxyl group one OH group. Accordingly, D-cHga-Pro-Arg-H is D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde and Bz-Ile-GluGly-Arg-pNA is benzoyl-L-isoleucyl -Lglutamil-glycyl-L-arginine-p-nitroanilide.

Coagulation is part of the body's defense mechanism. When a blood vessel is injured, the process starts and blood clots are formed to prevent bleeding. In addition, vascular disease, congestion in the blood, and abnormal activation of coagulation factors can cause coagulation. In this case, a blood clot (thrombus) is formed within the blood vessel, which partially or completely blocks it, thrombosis. Another part of the defense mechanism is fibrinolysis. The enzymes that work in it remove excess blood clots and are involved in thrombolysis and thrombus dissolution.

The process of blood coagulation is a series of catalyzed enzymatic reactions, a cascade reaction in which plasma proteins, so-called coagulation factors, are activated sequentially. Factors are denoted by Roman numerals and the active form is indicated by the letter. In some cases the trivial name (also) is used: for example, fibrinogen = factor I (fi), fibrin = factor Ia (son), prothrombin = factor II (phi) and thrombin = factor IIa (illa). During coagulation, serine proteases (fXIIa, fVIIa, fXIa, fIXa, fXa and thrombin), some accelerating cofactors (fVa and fVIIIa), and the clotting substance (fibrin) are produced. Of the resulting proteases, fXa and thrombin are the last two. FXa produces thrombin which cleaves fibrinogen to form a fibrin clot.

Previous mechanism of blood coagulation [R. G. MacFarlane, Naturre 202, 498 (1964); E. W. Davie and O. D. Ratnoff, Science 145, 1310 (1964)], fX is activated by two pathways, the so-called internal or external pathways. In the former case, a surface activated fXII (fXIIa) initiates the process by the fXI-> fXIa conversion followed by the flX-> fIXa conversion; fiX is activated by fIXa. Externally, the process begins with the appearance of a cell surface receptor, the tissue factor (TF), and the formation of the [fVIIxTF] and [fVIIa xTF] complex. Activation of fX is performed by the [fiVIla χ TF] complex.

According to recent results, coagulation in the living body is a combination of the above [E. W. Davie et al., Biochemistry 43: 10363 (1991)], and the most important steps are as follows.

1. In the event of an injury or disease of the vascular wall, TF is exposed to the surface and binds all of Factor VII in the blood. The resulting [fVIIxTF] complex is converted by a suitable protease (such as fXIIa, fXa, fIXa and thrombin) into the active [fVIIax TF] enzyme complex, which activates a small fraction of the plasma factor IX and X fIXa and fXa) and then inactivated by TFPI (Tissue Factor Pathway Inhibitor, formerly known as LACI (Lipoprotein-Associated Coagulation Inhibitor)), a common inhibitor of plasma fXa and [fVIIa χ TF] complex [T. J. Girard et al., Natur. 338, 518-520 (1989).

2. The resulting fIXa forms a so-called "tenase" complex with factor X and the fVIIIa accelerator molecule on the phospholipid (PL) surface in the presence of Ca ⁺⁺ :

[fIXa x fVIIIa xfXx PL xCa ⁺⁺ ] in which fX is activated, fXa is generated.

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3. The fXa formed so far with the prothrombin (fii) and the fVa accelerator molecule forms a so-called prothrombinase complex similar to tenase: [fXaxfVaxflIxPLxCa ⁺⁺ ]. This is where the prothrombin -> thrombin conversion takes place. The conversion of fV- »fVa and fVIII-> fVIIIa can be accomplished by fXa or thrombin.

4. The little thrombin produced converts a portion of fXI to fXIa and activates some of Factors VIII and V to produce new fVIIIa and fVa. Now, fXIa is converting factor IX to flXa. This can restart the chain reaction from the Xase complex to the formation of thrombin. As the process repeats itself, more and more thrombin is produced.

5. At sufficiently high thrombin concentrations, partial proteolysis of the fibrinogen dissolved in plasma occurs, the formation of the fibrin monomer, which is first associated with the so-called soluble fibrin polymer and then converted to the insoluble fibrin polymer. Thrombin also plays a role in the latter transformation, as it results in the formation of polymerization fXIIIa [L. Lorand and K. Konishi, Arch. Biochem. Biophys. 105, 58 (1964)].

The insoluble fibrin polymer is one of the major components of the blood clot and thrombus, and the other component is the platelet aggregate, which is also produced primarily by thrombin. The resulting thrombus or blood clot encapsulates a significant portion of the thrombin produced during the process, which initiates another clotting process when it is dissolved / dissolved in the thrombus [A. K. Gash et al., Am. J. Cardiol. 57: 175 (1986)]; R. Kumar et al., Thromb. Haemost. 72: 713 (1994)].

The foregoing highlights the key role of thrombin in thrombus formation. Therefore, substances that inhibit the function and / or production of thrombin are of particular importance in the treatment of thrombotic diseases.

Heparins and vitamin K antagonist coumarins (such as Syncumar and Warfarin), which indirectly inhibit thrombin, are currently the most widely and successfully used formulations for treating and preventing thrombosis.

Heparin catalyses the reaction between thrombin and its natural inhibitor, antithrombin III (AT-ΠΙ). However, this effect of heparin is absent when the plasma concentration of AT-III is less than 75% of normal [R. Egbring et al., Thromb. Haemost. 42, 225 (1979). Not least, this indirect mechanism cannot inhibit thrombin-bound thrombin because it is not accessible to the heparin-AT-III complex [J. I. Weitz et al., J. Clin. Invest. 86: 385 (1990)]. In addition, the incidence of treatment-induced bleeding complications and cases of thromboembolic events due to the immune process is not negligible (see, e.g., J.M. Walenga et al., Clin. Appl. Thrombosis / Hemostasis, 2 (Suppl. 1), S21-S27 (1996)).

Vitamin K antagonists may also be administered orally. They act after 16 to 24 hours and are shown to inhibit the formation of reactive Gla-containing forms of some clotting factors (such as prothrombin). The therapeutic effect requires partial inhibition (60-70%) [Μ. P. Esnouf and C. V. Prowse, Biochim. Biophys. Acta 490, 471 (1977)], which can be achieved by proper administration of the drug. The use of vitamin K antagonists is complicated by the narrow therapeutic band, the high dependence on the composition of the food (vitamin K) and the variable individual sensitivity.

The first high-potency synthetic substance directly inhibiting thrombin was D-Phe-Pro-Arg-H tripeptidaldehyde, which is a reversible inhibitor and showed significant anticoagulant activity both in vitro and in vivo [S. Bajusz et al., In: Peptides: Chemistry, Structure and Biology (R. Walter and J. Meienhofer, Eds.), Ann Arbor Publ., Ann Arbor, Michigan, USA, 1975, p. 603-608; Int. J. Peptide Protein Res. 12, 217 (1978)]. Several related compounds of D-Phe-Pro-Arg-H have been prepared. Boc-D-Phe-Pro-Arg-H [S. Bajusz et al., Int. J. Peptide Protein Slit. 12, 217 (1978)] and the chloromethyl ketone analogue (DPhe-Pro-Arg-CH ₂ Cl), which has been shown to be an irreversible inhibitor [C. Kettner and E. Shaw, Thromb. Gap. 14, 969 (1979)]. Among these, we highlight the boro-arginine analogs of the peptide and its acyl compounds (D-Phe and BocD-Phe, and Ac-D-Phe-Pro-boroArg), which are potent reversible inhibitors of thrombin [C. Kettner et al., J. Bioi. Chem. 265, 18289 (1990)] and other Boc-D-Phe-Pro-Arg-H analogs, including BocD-Chg-Pro-Arg-H analogue (PD Gesellchen and RT Shuman: EP 0 479 489 A2). (1992)].

D-Phe-Pro-Arg-H was found to be prone to spontaneous conversion in aqueous solution, but D-MePhe-Pro-Arg-H (GYKI-14,766) obtained by methylation of the terminal amino group was already sufficiently stable and similarly potent. Bajusz et al., U.S. Patent 4,703,036 (1987); J. Med. Chem. 33, 1729 (1990)]. It significantly inhibits coagulation and thrombus formation in experimental animals [D. Bagdy et al., Thromb. Haemost. 67, 357 and 68, 125 (1992); J. V. Jackson et al., J. Pharm. Ex. Ther. 261, 546 (1992)]; the inhibitory effect on fibrinolysis enzymes is insignificant, when co-administered with thrombolytics, it effectively promotes thrombus dissolution [C. V Jackson et al., J. Cardiovasc. Pharmacol. 21, 587 (1993)], which the heparin-AT-III complex is incapable of. Syntheses of D-MePhe-Pro-Arg-H related compounds have also been synthesized, for example, D-MePhg-ProArg-H [R. T. Shuman et al., 1993, J. Med. Chem. 36, 314].

Further stable and potent analogs of D-Phe-Pro-Arg-H were obtained by substituting the α-hydroxyacyl group for the terminal P-moiety. Some, such as the D-2-cyclohexyl-2-hydroxyacetyl analogue, D-Hma-Pro-Arg-H, exhibit high anticoagulant and antithrombotic activity similar to Cl. S. Bajusz et al., Bioorg. Med. Chem. 1995, 8, 1079].

The anticoagulant effect (inhibition of the proteolytic reactions of the process) is measured in coagulation tests, such as thrombin time (TT), activated partial thromboplast

HU 222,199 in Blin Time (APTT) and Prothrombin Time (PT) assays (EJW Bovie et al., Mayo Clinical Laboratory Manual of Hemostasis; WB Sanuders Co., Philadelphia, 1971). Plasma inhibited by spontaneous coagulation, such as citrate plasma, is induced to coagulate and the coagulation time is measured. The effect of anticoagulants is to prolong the coagulation time in proportion to the inhibition of the systemic reaction (s). For example, the anticoagulant effect is characterized by the concentration of the substance at which the coagulation time is doubled (IC ₅₀ ). The effect of anticoagulants on individual coagulation proteases is measured by the so-called amidolytic method [R. Lottenberg et al., Methods Enzymol. 80, 341 (1981); G. Cleason: Blood Coagulation and Fibrinolysis 5, 411 (1994)]. The isolated active factor (e.g., thrombin, fXa) and its chromogenic or fluorogenic peptide amide substrate are reacted in the absence or presence of the inhibitor. The enzyme inhibitory activity is characterized, for example, by the inhibition constant (IC ₅₀ ) as measured by amidolysis.

In the TT test, thrombin added to the citrate plasma causes coagulation. Systemic thrombin is 22 pmol / ml and its inhibition can be measured on the presence of fibrinogen (a natural substrate for thrombin) in the presence of plasma components. In the APTT and PT tests, the whole coagulation process takes place. Depending on the activator, fX is activated internally or externally. The resulting fXa activates prothrombin to thrombin, which eventually causes plasma clotting. Clotting time is prolonged when the inhibitor inhibits the enzymes in the assays or one of them. While up to 40 pmol / ml of Factor Xa can be produced in the APTT and PT assays (the amount of Factor X present in the two systems), thrombin may have a concentration of about 10 pmol / ml. 150 pmol / ml (APTT) and 350 pmol / ml (PT) are formed [cf. B. Kaiser et al., Thromb. Gap. 65: 157 (1992)].

In the case of D-MePhe-Pro-Arg-H (Cl), the double clotting times in the TT, APTT and PT tests were 87, 622 and 2915 nM, respectively. These values and the amount of thrombin present in the assays (22, 150 and 350 pmol / ml, respectively) increase similarly, suggesting that Cl behaves as a thrombin inhibitor in the APTT and PT assays and, for example, has little or no effect on the function of fXa. Consistent with this, Cl IC inhibits the amidolytic effect of thrombin on the Tos-Gly-Pro-Arg-pNA substrate at ₅₀ = 2 nM, while the amidolytic effect of fXa on the corresponding BzIle-Glu-Gly-Arg-pNA substrate is only slightly inhibited. _5O = 9.1 mM (S. Bajusz et al., Unpublished results).

It follows from the mechanism of blood coagulation that the process is inhibited not only by direct inhibitors of thrombin, but also by inhibitors of thrombin formation, such as fXa inhibitors. An example of a fXa inhibitor with significant anticoagulant and thrombus inhibiting activity is the 60-membered polypeptide isolated from the tick, Tick Anticoagulant Peptide [L. Waxman et al., Science 248, 593 (1990); Kelly et al., Circulation 86, 1411 (1992)] and DX-9065a (C2), a synthetic non-peptide: (+) - (2S) -2- [4 - [[(3S) -lacetimidoyl-3-pyrrolidinyl]. ] oxy] phenyl] -3- [7-amidino-2-naphthyl] -propionic acid hydrochloride pentahydrate [T. Hara et al., Thromb. Haemost. 71, 314 (1994); T. Yokoyama et al., Circulation 92, 485 (1995)]. These substances strongly inhibit the amidolytic activity and plasma clotting of fXa in the PT and APTT assays. That is, they inhibit both free and complex factor Xa well. However, as specific fXa inhibitors, they do not inhibit thrombin and do not work in the TT assay. Examples of the fXa synthetic peptide inhibitor are 4MeP-Asp-Pro-Arg-H (C3) (International Publication No. WO 93/15756) and Boc-D-PheNal (1) -Arg-H (C4) (WO 95 (International Application Publication No. 13693). C3 and C4 are reported to inhibit the amidolytic effect of fXa on ZD-Arg-Gly-Arg-pNA substrate at ₅₀ = 57 and 30 nM; no data are available on their anticoagulant activity. In our own experiments, BzIle-Glu-Gly-Arg-pNA substrate also showed significant inhibitory activity on C3 and C4, while their anticoagulant activity in plasma coagulation assays is extremely low. Table 1 shows the (C2) 'and (C3-C4) activity values reported for the above synthetic fXa inhibitors as compared to the Cl anti-thrombotic data.

The data in Table 1 show that the anticoagulant effect observed in the PT and APTT assays is due to inhibition of thrombin in Cl and inhibition of fXa in C2. However, the significant fXa inhibitory activity of C3 and C4 is not accompanied by significant anticoagulant activity. It is likely that C3 and C4 do not have access to the active center of fXa in the prothrombinase complex, these peptides being able to inhibit only the free, soluble factor Xa.

Table 1

Effects of known synthetic inhibitors Factor Xa (A) and anticoagulant (B)

THE: B: IC ₅₀ , μΜ ' inhibitor IC ₅₀ , nM ^b PT APTT TT cl 9133 2.91 0.62 0.09 C2 70 0.52 0.97 NA ^C C3 64 19.32 4.59 0.87 C4 86 53.62 9.96 17,24

^the peptide concentration at which the clotting time of the control goes to double the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) test.

^b Measured on isolated Bz-Ile-Glu-Gly-Arg-pNA chromogenic substrate with human factor Xa. ^c NA = not active.

According to another announcement, [N. A. Prager et al., Circulation 92, 962 (1995)], not only thrombin but also factor Xa, which are trapped in the thrombus / blood clot and released during dissolution, contribute to the initiation and maintenance of a new coagulation process, e.g. fVII χ TF] complex and activation of factors V and VIII. Thus, it is preferred that anticoagulants also inhibit factor Xa in addition to thrombin

It is particularly advantageous if this inhibitory effect extends to coagulated thrombin and factor Xa.

It is an object of the present invention to provide novel peptide derivatives which inhibit the coagulation process more than is known and exhibit an inhibitory effect when administered orally.

It has been observed that the known αD-hydroxyacyl-Lprolyl-L-arginine aldehydes [S. Bajusz et al., Bioorg. Med. Chem., 8: 1079 (1995)] of about 30-fold lower IC inhibit factor Xa amidolytic effect as CI value _50, which show a similar significant anticoagulant activity. For example, D-2-cyclohexyl-2-hydroxyacetyl analogue, D-Hma-Pro-Arg-H (C5). It has now surprisingly been found that the analogues obtained with 2-cycloheptyl-2-hydroxyacetic acid and 2-cyclopentyl-2-hydroxyacetic acid, D-cHga and D-cPga-ProArg-H, further inhibit factor Xa. , and still exhibit significant anticoagulant activity.

Accordingly, the present invention provides novel peptide derivatives of formula (I): D-Xaa-Pro-Arg-H (I) wherein:

Xaa is 2-cycloheptyl-2-hydroxyacetyl or 2-cyclopentyl-2-hydroxyacetyl,

Pro is an L-proline residue and

Arg is the L-arginine residue and its acid addition salts with an organic or inorganic acid, and pharmaceutical compositions containing the above compounds.

The compounds of formula (I) of the present invention wherein Xaa, Pro and Arg are as defined above are prepared, for example, by condensation of a QD-Xaa-Pro acyl dipeptide protected on the OH group with a Q protecting group on benzyloxycarbonyl. , the resulting protected peptide lactam is reduced to the protected peptide aldehyde QD-Xaa-ProArg (Z) -H, and finally the Z group is removed from the guanidone group of arginine and the Q group is removed from the α-hydroxy group, and isolating the peptide derivative of formula (I) in the form of an organic or inorganic acid addition salt.

For example, the starting compound QD-Xaa-Pro acyl dipeptide is prepared by converting the corresponding D-Xaa alpha-hydroxy acid into a benzyl ester, protecting the OH group with Q protecting group and then freeing the resulting QD-Xaa-OBzl by hydrogenolysis. acid and coupled with L-proline benzyl ester. The required O-protected acyl dipeptide is obtained after removal of the benzyl ester by hydrogenolysis.

Preferably, the OH-protected Q-DXaa of Q-DXaa required for coupling to L-proline can be prepared by O-acetylation of the DL-Xaa racemic compound followed by removal of the acetyl group from Ac-D-Xaa by enzymatic hydrolysis. . Preferably, the racemic compound DLXaa can be resolved with D-tyrosine hydrazide.

The compounds of the formula I in which Xaa, Pro and Arg are as defined above have potent anticoagulant activity both in vitro and in vivo, with favorable bioavailability.

The in vitro anticoagulant activity of the compounds of Formula I was determined in the Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and Thrombin Time (TT) assays [D. Bagdy et al., Thromb. Haemost. 67: 325 (1992)]. The factor Xa inhibitory activity of the compounds on the Bz-Ile-Glu-Gly-Arg-pNA chromogenic substrate was also determined (see Method M6).

The results obtained are shown in Table 2; Corresponding data for Cl and C5 are used as controls. The table lists the compounds in descending order of PT activity. The data show a beneficial influence of the terminal cHga and cPga moieties over the Hma and MePhe moieties.

Table 2

Effect of the novel peptidyl-arginine aldehydes and control compounds of similar structure on anticoagulant (A) and factor Xa (B) inhibitory activity in descending order of PT activity

A: IC ₅₀ , μΜ ' B: IC ₅₀ Peptidyl Arginine Aldehyde (Number ^b ) PT APTT TT nM <* D-cHga-Pro-Arg-H (J) 1 1.20 0.37 0.11 63 D-cPga-Pro-Arg-H (2) 2.03 0.71 0.10 107 Compounds control D-Hma-Pro-Arg-H (C5) 2.14 0.79 0.22 247 D-MePhe-Pro-Arg-H (Cl) 2.92 0.62 0.09 9133

^the concentration of peptide at which the clotting time is doubled.

^b Identical to the number of the exemplifying embodiment of Compound a, or Control Compound (Cl, C5).

^{d The} value measured with Bz-IIe-Glu-Gly-Arg-pNA substrate isolated with human factor Xa (see Method M1-M5).

Recombination of citrate plasma B1 and B1 fibrinogen was obtained by hanging human fibrinogen with human thrombin, and the ZD-Arg-Gly-Arg-pNA and Tos-Gly-Pro5 Arg-pNA substrate were used to measure the activity of factor Xa and thrombin (see M1 -M5 method).

The plasma-clotting factor Xa and thrombin inhibitory activity of the compounds of formula I and the fibrin gel-bound thrombin inhibitory activity are shown in Table 3 for the example of compound 7; the corresponding values for Cl and C5 are shown as controls. Plasma clot is lamellar human

Table 3

Inhibitory effect (IC ₅₀ , μΜ) of the novel peptidyl-arginine aldehyde (1) and control compounds (Cl, C5) of the invention on plasma coagulated factor Xa and thrombin and fibrin gel-bound thrombin ⁸

Plazmaalvadék fibrin Peptidyl Arginine Aldehyde (Number ^B ) Factor Xa thrombin thrombin D-cHga-Pro-Arg-H (1) 0.20 0.52 0.21 Compounds control D-MePhe-Pro-Arg-H (Cl) 1.12 0.38 0.30 D-Hma-Pro-Arg-H (C5) 0.19 0.27 0.22

^the determination of IC ₅₀ values the activity of factor Xa and thrombin was measured Ml -M5 Moc-D-Chg-Gly-Arg-pNA or Tos-Gly-Pro-Arg-pNA substrate. methods.

^b Identical to the number of the exemplifying embodiment of the new compound and to the control compound (Cl and C5).

These data show that compound of formula (1) according to the invention in a closed factor Xa and human thrombin lemezkedús in plasma and thrombin bound to human fibrin magnitude below both micromoles / nanomolar IC ₅₀ value can inhibit, resemblance 30 Loan C5.

The inhibitory effect of the novel compounds of the invention on plasmin (PL) and tissue plasminogen activator (tPA) and urokinase (UK) -induced plasma formation was investigated by the fibrin plate method [D. 35 Bagdy et al., Thromb. Haemost. 67: 325 (1992)]. Cl and C3 were used as controls. Of these, the inhibitory effect of Cl on mock fibrinolysis was insignificant in vivo, it was useful as adjuvant in dissolving the experimental thrombus, and the inhibitory effect of C3 on fibrinolysis was demonstrated in vivo [C. V. Jackson et al., J. Cardiovasc. Pharmacol. 21, 587 (1993)].

By way of example, the results obtained with the new compounds 7 and 2 and the control Cl and C3 are shown (Table 4). In addition to IC ₅₀ values (columns A), the relative potency of the compounds with respect to C7 (B columns) is also indicated. The latter indicate that the new 7 and 2 inhibitory effects on fibrinolysis are similar to those of Compound CI and are 7.5-12-39 times less active than Compound C3 on the three enzymes tested.

Table 4

Inhibitory effect (IC ₅₀ ) of novel peptidyl-arginine aldehydes and control compounds of the present invention on plasmin (PL) and tissue plasminogen activator (tPA) and urokinase (UK) -induced plasma fibrin plate ⁸

A: IC ₅₀ and B: relative efficiency ¹¹ E.G PA UK Peptidyl Arginine Aldehyde (Number ^c ) THE B THE B THE B D-cHga-Pro-Arg-H (1) 83 0.6 74 1.8 120 0.7 D-cPga-Pro-Arg-H (2) 3.9 0.13 112 1.2 137 0.6 D-MePhe-Pro-Arg-H (Cl) 54 1.0 132 1.0 82 1.0 Boc-D-Phe-Pro-Arg-H (C3) 12 4.5 6 22.0 3 27.3

^the IC 50 = peptide concentration (mM) at which the fibrin-hydrolyzed area reduced to 50% of control. ^b Values relative to Cl efficiency (1 / IC50 = 1).

^c Identical to the number of the exemplifying embodiment of the new compound and to the control compound (Cl and C3).

HU 222 199 Β1

The anticoagulant and platelet aggregation inhibitory activity of the compounds of formula (I) was tested ex vivo on New Zealand white rabbits [D. Bagdy et al., Thromb. Haemost. 67: 357 (1992)]. The compounds were dissolved in buffered saline by intravenous injection (0.04-5.0 mg / kg) or infusion (0.25-5.0 mg / kg / h) or 0.5-6.0 mg / kg. subcutaneously or at a dose of 2.5 to 20 mg / kg orally. The effect of the compounds is detectable <30 minutes after oral administration, with peak values of 60-180. and the therapeutic effect is maintained in a dose-dependent manner for 3 to 6 hours.

The in vivo activity of D-cHga-Pro-Arg-H (1) is shown in detail in Tables 5 and 6; the corresponding values for Cl are shown as controls. The compound was administered orally at a dose of 5 mg / kg. Blood samples were taken every 30-60 minutes from the animal's stomach. Whole blood coagulation time (WBCT) and the degree of inhibition of thrombin-induced platelet aggregation (PAI) were determined. In addition, the activated partial throm10 boplastin time (APTT) and thrombin time (TT) of citrate plasma from the blood sample were determined. APTT and TT are shown in Table 5, and WBCT and PAI are shown in Table 6.

Table 5

Anticoagulant effect of D-cHga-Pro-Arg-H and D-MePhe-Pro-Arg-H in rabbits at 5 mg / kg oral dose, characterized by relative coagulation times in the APTT and TT test ³

D-cHga-Pro-Arg-H (1) D-MePhe-Pro-Arg-H (Cl) Time (p) APTT TT APTT TT 0 1.0 1.0 1.0 1.0 30 2.01 ± 0.43 10.57 ± 5.98 1.27 + 0.38 1.52 & 0.17 45 2.25 ± 0.43 10.89 ± 5.89 1.30 + 0.02 2.50 + 0.44 60 2.32 ± 0.43 11.67 ± 5.69 1.37 + 0.03 4.75 ± 1.38 90 2, ll ± 0.31 14.00 ± 6.48 1.45 + 0.09 10.50 ± 5.49 120 l, 84 ± 0, II 10.60 ± 4.22 1.43 & 0.11 5.51 ± 3.59 180 l, 66 ± 0.10 2.22 & 0.71 1.39 + 0.09 2.05 + 0.38 240 1.37 + 0.08 1.23 + 0.07 1.31 & 0.11 1.81 + 0.39 300 1.31 + 0.09 1.13 + 0.05 1.25 + 0.22 -

³ Ratio of coagulation times in treated and untreated animals. Therapeutic values were highlighted.

Table 6

Inhibition of coagulation and platelet aggregation (PAI) of D-cHga-Pro-Arg-H and D-MePhe-Pro-Arg-H in rabbits at 5 mg / kg oral dose with relative clotting time and% characterized by inhibition ³

D-cHga-Pro-Arg-H (1) D-MePhe-Pro-Arg-H (Cl) Work (p) WBCT PAI (%) WBCT PAI (%) 0 1.0 0 1.0 0 30 l, 80 ± 0.36 71.0 ± 19.4 1.07 + 0.13 52.8 ± 14.7 45 2.03 ± 0.35 74.0 ± 13.9 1.26 & 0.07 58.4 ± 15.6 60 l, 98 ± 0.30 91.0 ± 4.5 l, 55 ± 0.17 54.2 ± 11.6 90 l, 78 ± 0.26 93.0 ± 3.5 l, 61 ± 0.34 83.2 ± 8.9 120 l, 52 ± 0.13 92.4 ± 6.5 1.45 + 0.21 75.2 ± 12.3 180 1.29 & 0.07 71.6 ± 19.8 1.44 & 0.08 47.5 20.9 + 240 1.13 + 0.04 49.4 21.5 + 1.22 + 0.08 32.2 + 16.0

^a Ratio of coagulation times in treated and untreated animals. Therapeutic values were highlighted.

HU 222 199 Bl

The data in the tables indicate that the new compound 1 has a greater and more lasting effect on the anticoagulant (anti-coagulant) and anti-platelet aggregation than the control Cl.

The compounds of the invention (I) are used for the treatment and prevention of the following conditions associated with thrombosis and / or hypercoagulability: deep vein thrombosis, pulmonary embolism, arterial thrombosis, unstable angina, myocardial death, atrial fibrillation and thrombosis-based strokes. They may also be used in the prevention of thrombotic diseases of the coronary artery and cerebral artery in the case of atherosclerosis, for the surgical prophylaxis of high-risk patients and for other surgical prophylaxis. In addition, they are useful in the prevention of thrombolysis or percutaneous transluminal angioplasty, and in the treatment of hemodialysis and adjunctive therapy in nephrosis syndrome, and in the treatment of diseases such as malignant neoplasms, inflammation, and inflammation. They may also be used in cases where the administration of other anticoagulants is ineffective or contraindicated: for example, in the case of heparin, lack of antithrombin-III or HIT (for heparin-induced thrombocytopenia), for example, in the case of coumarins, gravity.

For therapeutic purposes, the compounds of the present invention and their pharmaceutically acceptable salts may be used alone or conveniently in the form of a pharmaceutical composition. Such compositions are also included within the scope of the present invention.

These pharmaceutical compositions contain as an active ingredient an amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof in association with pharmaceutically acceptable carriers, excipients, diluents and / or other pharmaceutical auxiliaries which are customary in the art.

Suitable carriers, diluents or fillers as described above include water, alcohols, gelatin, lactose, sucrose, starch, pectin, magnesium stearate, stearic acid, talc, various animal or vegetable oils, and glycols such as propylene glycol or polyethylene glycol. The pharmaceutical excipients include preservatives, various natural or synthetic emulsifying, dispersing or wetting agents, coloring agents, flavoring agents, buffering agents, disintegrating agents, and other agents that promote the bioavailability of the active ingredient.

The pharmaceutical compositions of the invention may be in conventional forms. Examples of such common dosage forms include oral (oral) formulations (tablets, capsules, powders, pills, dragees or granules) and parenteral (gastro-intestinal by-products), suppository, patch or ointment).

Although the dose of the compounds of the present invention required to exert a therapeutic effect will vary depending upon the individual condition and age of the patients being treated, it may vary and will ultimately be determined by the attending physician; for the prevention and / or treatment of diseases in which inhibition of the function and / or production of thrombin is desirable, generally from about 0.01 mg to about 1000 mg and preferably from 0.25 mg to 20 mg per kg of body weight of these compounds. daily oral or parenteral, for example, intravenous doses.

When administered in combination with thrombolytics (e.g., tPA or urokinase), the compounds of formula (I) of the present invention can effectively promote dissolution of thrombi formed in arteries or veins or effectively inhibit thrombus re-formation. In such cases, the compounds of the present invention may be administered concurrently with or after thrombolytic therapy.

The invention is illustrated by the following non-limiting examples.

In these examples, _Rf values were determined by thin layer chromatography on silica gel (DC-Alufolien Kieselgel 60 F ₂₅ 4, Merck, Darmstadt) in the following solvents:

1. Ethyl acetate

2. Ethyl acetate-pyridine-acetic acid-water (480: 20: 6: 11)

3. Ethyl acetate-pyridine-acetic acid-water (60: 20: 6: 11)

6. Ethyl acetate-pyridine-acetic acid-water (240: 20: 6: 11)

12. Ethyl acetate-cyclohexane (1: 9)

14. Chloroform-acetic acid (95: 5)

The capacity factor values (k ') in the examples were determined using a Pharmacia LKB analytical HPLC System Two as follows.

Column: "VYDAC C-18 reversed phase: 10 pm, 300,, 4 χ 250 mm".

Buffer A: 0.1% trifluoroacetic acid in water.

Buffer B: 0.1% trifluoroacetic acid in acetonitrile.

The gradient used was a flow rate of 1 ml / min

I: 0-5 min 0-25% B, then isocratic 25% B;

II: 0-30 min 0-60% B.

For HPLC analysis, the gradient used (I or II) is given in brackets after the truncation in each step of the example.

The optical purity values given in the examples were determined by the above-mentioned HPLC apparatus and are indicated by the term "chiral HPLC" in parentheses.

Column: Chiralpack WH (DAICEL) 4x250 mm

Eluent: 0.25 mM CuSO ₄ ; flow rate 1 ml / min.

The analyzes were performed at 50 ° C.

The peptide content of the eluate was detected by UV light at 214 nm. The concentration of the sample is 1 mg / ml buffer A (reverse phase) and methanol (chiral phase), the injection volume is 25 µl.

Acyl-arginine aldehydes exist in equilibrium forms: aldehyde and aldehyde hydrate, and two aminocyclic forms.

HU 222 199 BI

Of these, the aldehyde hydrate and one or both of the aminocyclols appear in separate peaks during HPLC. Accordingly, acyl-arginine aldehydes are characterized by two or three k 'values found in the examples.

Spectroscopy. FAB positive ionisation measurements were performed on a Finnigan MÁT 8430 instrument. Samples were dissolved in a m-nitrobenzyl alcohol matrix and directly introduced into the ion spinning. In addition to the molecular ion in the spectrum of peptidyl-arginine aldehydes, the molecular ion of the addition compound formed with m-nitrobenzyl alcohol (NBA) is also shown: [M + H] + and [M + H + NBA] ⁺ . Accordingly, FAB mass spectral data are given in the examples. ESI positive ionization measurements were performed on a VG Quattro (Fisons). Samples were dissolved in 1: 1 acetonitrile / water / v / v formic acid and flowed to the ion source using a 10 mL sample loop at a flow rate of 15-25 mL / min.

Specific rotation values ([α] _D ) were measured at 20 ° C.

Example 1

Preparation of D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-Larginine aldehyde (D-cHga-Pro-Arg-H) hemisulfate

Step 1: Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam

Tert-butyloxycarbonyl-N ^G -benzyloxy-carbonyl-L-arginine lactam 7.85 g (20.1 mmol) [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] are suspended in 20 ml of chloroform, and 20 ml of hydrochloric acid ethyl acetate (0.11-0.15 g / ml) are added under stirring and ice-cooling. The cleavage of the Boc group is followed by layer chromatography [Rj (3) = 0.5 (free compound); 1.0 (Boc compound)]. At the end of the reaction, the slurry was diluted with diethyl ether (40 mL), the precipitated crystalline material was filtered off, washed with acetone (10 mL) and diethyl ether (10 mL) and dried in a vacuum desiccator over potassium hydroxide for about two hours. The resulting N ^G -benzyloxycarbonyl-L-arginine-lactam chlorohydrate was dissolved in 20 ml of dimethylformamide, cooled to -20 ° C and added to the following mixed anhydride.

Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-proline (7.1 g, 20.1 mmol) (Example 1, Step I) was dissolved in 20 mL of dimethylformamide, cooled to -20 ° C. and treated with 2.23 ml (20.1 mmol) N-methylmorpholine and 2.65 ml (20.1 mmol) of isobutyl chloro-ester, followed by 10 minutes stirring, N ^G -benzyloxy-carbonyl stirring A solution of the above in dimethylformamide containing L-arginine lactam and finally enough triethylamine to bring the reaction mixture to a pH of about 8 (about 2.8 mL required). The reaction mixture was stirred for 30 minutes at -10 ° C and then for 1 hour at 0 ° C. The salts were filtered off and the filtrate was diluted with 100 mL of ethyl acetate. The resulting solution was washed with water (3 x 25 ml), 1M potassium bisulfate (10 ml) and water (3 x 10 ml), and after drying over anhydrous sodium sulfate, concentrated under a pressure of 2.0-2.5 kPa. The product was chromatographed on a silica gel column (200 g, Kieselgel 60) using ethyl acetate as eluent. The pure fractions [Rf (1) = 0.60] were combined and evaporated under a pressure of 2.0-2.5 kPa. The residue was crystallized with diisopropyl ether. 8.1 g (64%) of the title compound are obtained. Rf (l) = 0.6. Melting point: 66-68 ° C. The FAB mass spectrum (626 [M + H] ⁺ , 779 [M + H + NBA] ⁺ ) confirms the expected structure.

Step 2: Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde

8.0 g (12.8 mmole) of tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-Arginine ar-lactam (Example 1, first Step 1) is dissolved in 15 ml of tetrahydrofuran and 3.6 mmol of lithium aluminum hydride dissolved in tetrahydrofuran is added with stirring at -50 ° C. The progress of the reduction is monitored by layer chromatography in ethyl acetate-pyridine-acetic acid-water (240: 20: 6: 11) and additional lithium aluminum hydride is added as needed. To the reaction mixture was added dropwise cooled 0.5 M sulfuric acid to pH 3 with cooling and stirring and diluted with water (35 mL). The resulting solution was extracted with n-hexane (2 x 15 mL) and extracted with methylene chloride (3 x 20 mL). The methylene chloride solutions were combined, washed with water (3 x 15 mL), cold 5% sodium bicarbonate (15 mL) and water (15 mL), and then, after drying over anhydrous sodium sulfate, concentrated under a pressure of 2.0-2.5 kPa. The residue was triturated with diisopropyl ether, filtered, and dried in a vacuum desiccator. 7.25 g (90%) of the title product are obtained. Rf (6) = 0.40. Mp 107 ° C. [α] _D = + 16.0 ° (c = 1, in tetrahydrofuran). The FAB mass spectrum (628 [M + H] ⁺ , 781 [M + H + NBA] ⁺ ) confirms the expected structure.

Step 3: D-2-Cycloheptyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde hemisulfate

Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-Arginine aldehyde ar (7.05 g, 11.23 mmol) (Example 1, 2nd Step 1) is dissolved in 60 ml of ethanol, 11.23 ml of 0.5 M sulfuric acid and 13.3 ml of water are added, followed by 0.7 g of palladium on charcoal catalyst suspended in 25 ml of ethanol and ca. Hydrogenate at 10 ° C. The reaction, followed by layer chromatography, is complete within about 15 minutes. The catalyst was filtered off and the solution was pressurized to about 2.0-2.5 kPa. Concentrate to 7-9 ml. The residue was diluted with water (80 ml), extracted with methylene chloride (4 x 15 ml) and the aqueous solution was allowed to stand for 24 hours at 20-22 ° C. The solution was shaken again with methylene chloride (3 x 15 mL), adjusted to pH 3.5 with Dowex AG 1-X8 ion exchange resin, then frozen and lyophilized. 4.28 g (83%) of the title compound are obtained. [α] _D = -94.7 ° (c = 1; in water). HPLC (I): k '= 4.93 and 5.25. The FAB mass spectrum (410 [M + H] +, 563 [M + H + NBA] ⁺ ) confirms the expected structure.

For example, the starting material is prepared as follows:

HU 222 199 Bl

Preparation of Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-proline (THP-D-cHga-Pro)

Step A: Dicyclohexylammonium salt of O-acetyl-DL-2-cycloheptyl-2-hydroxyacetic acid

1.72 g (10 mmol) of DL-2-cycloheptyl-2-hydroxy-acetic acid [H. Takeshita et al., Bull. Chem. Soc. Japan 47, 1767 (1974)] was dissolved in 20 ml of anhydrous pyridine, 9.4 ml (100 mmol) of acetic anhydride was added and the solution was allowed to stand at room temperature for 24 hours and then concentrated at 2.0-2.5 kPa. and the residue was dissolved in 25 ml of diethyl ether. The solution was washed with water, 1M potassium bisulfate, water again, and then dried over anhydrous sodium sulfate. The residue was dissolved in diethyl ether (5 mL), dicyclohexylamine (2.1 mL, 10.5 mmol) and n-hexane (20 mL) were added. The solution was allowed to stand in the refrigerator for 2 days. The precipitated crystals were filtered off, washed with 3 x 10 ml of n-hexane and air-dried. 2.56 g (64.6%) of the title product are obtained.

Analysis for C ₂₃ H ₄₁ NO ₄ (395.57):

Found: C, 69.83; H = 10.45%; % N, 3.54; Found: C, 69.9; H, 10.7; N, 3.5.

Step B: Resolution of 0-acetyl-DL-2-cycloheptyl-2-hydroxy-acetic acid with acylase I

Dicyclohexylammonium O-acetyl-DL-2-cycloheptyl-2-hydroxyacetic acid (1.58 g, 4.0 mmol) was dissolved in diethyl ether (15 mL) and 1M potassium hydrogen sulfate (5 mL). The phases are separated, the diethyl ether phase is washed with water to neutral and then dried over sodium sulfate. The residual oil was 0.86 g (4.0 mmol) of O-acetyl-DL-2-cycloheptyl-2-hydroxyacetic acid, which was dissolved in 20 mL of 0.2 M sodium bicarbonate and 0.01 g of cobalt was added. (II) chloride hexahydrate and 0.005 mg Acylase I (Sigma, 2000-3000 units / mg) and allowed to stand for approx. At 25 ° for 3 days. The aqueous solution was acidified with 4.5 mL of 1 M potassium bisulfate and extracted with 3 x 10 mL of diethyl ether. The diethyl ether layers were combined, washed with water and then dried over anhydrous sodium sulfate. The residual oil is L-2-cycloheptyl-2-hydroxy-acetic acid [Rf (14) = 0.2] and O-acetyl-D-2-cycloheptyl-2-hydroxy-acetic acid [Rf (14) ) = 0.5], which was chromatographed on a silica gel column using chloroform-acetic acid (95: 5). The pure product fractions were combined and concentrated at 2.0-2.5 kPa.

The residue from evaporation 1 [Rf (14) = 0.5] is 0.34 g (79%) of an oil, O-acetyl-D-2-cycloheptyl-2-hydroxy-acetic acid, which is used directly in the next step C. .

Evaporation residue 2 [Rj (14) = 0.2] was 0.25 g (80%) of a solid, L-2-cycloheptyl-2-hydroxyacetic acid, which was filtered with n-hexane and air dried. Melting point: 78 ° C. [α] _D = + 18.15 ° (c = 1, methanol). Optical purity = approx. 95% (chiral HPLC).

Step C: D-2-Cycloheptyl-2-hydroxy-acetic acid (Na-methylate deacetylation of the Oacetyl derivative)

The O-acetyl-D-2-cycloheptyl-2-hydroxyacetic acid obtained in the previous step B (evaporation residue 1) was dissolved in methanol (1.59 mmol) containing sodium methylate. The solution was allowed to stand for 24 hours and then concentrated. The residue was dissolved in 10 ml of diethyl ether and 2.5 ml of 0.5 M potassium bisulfate, the diethyl ether layer was washed with water, dried over anhydrous sodium sulfate and evaporated. The crystalline residue was triturated with hexane, filtered and air dried. Yield: 0.21 g (61%). [α] _D = -14.7 ° (c = 1 in methanol). Optical purity = approx. 86% (chiral HPLC).

Step D: D-2-Cycloheptyl-2-hydroxy-acetic acid (Resolution of DL with D-tyrosine hydrazide

DL-2-cycloheptyl-2-hydroxyacetic acid (17.2 g, 100 mmol) and D-tyrosine hydrazide (19.52 g, 100 mmol) were suspended in anhydrous ethanol (600 mL) and heated to reflux until completely dissolved. After the solution has cooled to room temperature, it is kept in the refrigerator overnight. The precipitated crystals are filtered off, washed with 2 x 20 ml of cold anhydrous ethanol and dried in a vacuum desiccator. The diastereomeric salt (19.54 g, 53.18 mmol) was recrystallized twice from 15.3 ml / g of anhydrous ethanol. 12.45 g (67.7%) of the diastereomeric salt are obtained, which is dissolved in 50 ml of diethyl ether and 50 ml of 1 M potassium hydrogen sulfate. The diethyl ether layer was washed with water to anhydrous, dried over anhydrous sodium sulfate and then concentrated under a pressure of 2.0-2.5 kPa. The crystalline residue was triturated with n-hexane, filtered and air dried. 5.61 g (32.57 mmol, 65.2%) of D-2-cycloheptyl-2-hydroxyacetic acid are obtained. Melting point: 80 ° C. [α] _D = -20.2 ° (c = 1; in methanol) and -30 ° (c = 1; in acetic acid). Optical purity => 98% (chiral HPLC).

Analysis for C9 ₁₆ O ₃ (172.22) Calcd:

Calculated: C, 62.76; % H, 9.36;

Found: C, 62.01; H% = 9.31.

Step E: D-2-Cycloheptyl-2-hydroxy-acetic acid benzyl ester

D-2-cycloheptyl-2-hydroxyacetic acid (5.51 g, 32 mmol) (Example 1, Step D) was dissolved in dimethylformamide (37 mL), benzyl bromide (3.7 mL, 31.3 mmol) was added and Dicyclohexylamine (6.34 mL, 32 mmol) was added and the mixture was stirred at room temperature for 24 hours. The reaction mixture was filtered and concentrated at 2.0-2.5 kPa. The residue was dissolved in 50 ml of diethyl ether and 50 ml of 0.5 M potassium hydrogen sulfate. The diethyl ether phase was washed with water anhydrous and then washed with 2 x 10 mL of 5% sodium bicarbonate and 2 x 10 mL of water. It is then dried over anhydrous sodium sulfate and concentrated under a pressure of 2.0-2.5 kPa. 7.4 g (88%) of the title compound are obtained in the form of an oil. R (12) = 0.20. [α] _D = + 13.2 ° (c = 1, in methanol).

Step F: Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxy-acetic acid benzyl ester

D-Cycloheptyl-2-hydroxyacetic acid benzyl ester (7.4 g, 28 mmol) (Example 1, Step E) was dissolved in methylene chloride (65 mL) and treated with 3.6 mL (39.4 mmol) 2, 4-dihydropyran and 0.3 mL of hydrochloric acid in ethyl acetate (concentration 0.11-0.15 g / mL) and allowed to stand at room temperature for 16 hours. The reaction mixture was diluted with methylene chloride (40 mL), washed with water (3 x 20 mL),

It is dried over 20 ml of cold 5% sodium bicarbonate, again with 2 x 20 ml of water, then dried over anhydrous sodium sulphate and concentrated under a pressure of 2.0 to 2.5 kPa. The resulting evaporation residue [R _f (12) = 0.30] is considered to be 28 mmol of the title product.

Step G: Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxy-acetic acid Benzyl ester of tetrahydropyranyl-D-cycloheptyl-2-hydroxy-acetic acid (Example 1, Step F) was dissolved in methanol (50 mL) and charcoal (0.1 g). hydrogenation with palladium catalyst. The reaction was monitored by chromatography [Rf (12) = 0.30 (ester); 0.00 (self); R ((2) = 1.00 (ester); 0.8 (acid)] At the end of the reaction, the catalyst was filtered off, the filtrate was evaporated under a pressure of 2.0-2.5 kPa and the residual oil was dried in a vacuum desiccator. Obtained as an oil (7 g, 25 mmol, 89%) Rf (2) = 0.80.

Step H: Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-prolylbenzyl ester mmol of tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetic acid (Example 1, Step G) was dissolved in 25 mL of dimethyl formamide and 3.83 g (25 mmol) of 1-hydroxybenzotriazole hydrate, 2.8 ml (25 mmol) of N-methylmorpholine and 6.04 g (25 mmol) of L-proline benzyl- ester hydrochloride followed by ice-cooling and stirring, 5.16 g (25 mmol) of dicyclohexylcarbodiimide. The reaction mixture was stirred at 0 ° C for 1 hour and at room temperature overnight, then filtered and concentrated under a pressure of 2.0-2.5 kPa. The residue was dissolved in diethyl ether (100 mL) and washed with 5% sodium bicarbonate (3 x 20 mL), water, 1M potassium bisulfate and water, then dried over anhydrous sodium sulfate and evaporated. The evaporation residue was dried in a vacuum desiccator. Obtained as an oil (9.9 g, 90%). Rf (L) = 0.80.

Step I: Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-proline

Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-proline benzyl ester (9.9 g, 22.5 mmol) (Example 1, Step H) was dissolved in methanol (100 mL) and charcoal (0.1 g). hydrogenation with palladium catalyst. The progress of the reaction is monitored by chromatography [Rf (l) = 0.80 (ester); 0.00 (self); R 1 (2) = 1.00 (ester); 0.30 (acid)]. At the end of the reaction, the catalyst is filtered off, the filtrate is evaporated to a pressure of 2.0-2.5 kPa and the residual oil is dried in a vacuum desiccator. 7.2 g (90%) of the title compound are obtained in the form of an oil [R] (2) = 0.30], the major portion of which is used directly in Step 1 of the Example and the smaller portion is converted to the crystalline salt as follows.

Tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-proline (0.35 g, 1 mmol) was dissolved in diethyl ether (5 mL) and cyclohexylamine (0.115 mL, 1.05 mmol) was added. It was washed with ether and dried in a vacuum desiccator. 0.40 g (90%) of tetrahydropyranyl-D-2-cycloheptyl-2-hydroxyacetyl-L-proline-cyclohexylammonium salt are obtained. Melting point: 146-150 ° C. [α] _D = -12.8 ° (c = 1, in ethanol). Analysis for C25H44N2O ₅ (452.62) Calcd:

Found: C, 66.34; % H, 9.80; % N, 6.19; Found: C, 66.2; % H, 9.90; % N, 6.08.

Example 2

Preparation of D-2-cyclopentyl-2-hydroxyacetyl-L-prolyl-L-arginaldehyde (D-cPga-Pro-Arg-H) Hemisulfate

Step 1: Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine lactam

0.82 g (2.1 mmol) of t-butoxycarbonyl-N ^G -benzyloxycarbonyl-L-arginine lactam [S. Bajusz et al., J. Med. Chem. 33, 1729 (1990)] and 0.68 g (2.1 mmol) of tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-proline (Example 2, G). Step 1) Starting as described in Example 1, Step 1, coupling of the components and chromatographic purification of the final product using proportional amounts of reagents and solvents. The pure fractions containing the final product [Rf (1) = 0.4] were combined and evaporated to a pressure of 2.0-2.5 kPa and the residue was dried in a desiccator. The resulting oil (0.59 g, 47%) was considered as the title product (0.97 mmol). The FAB mass spectrum (598 [M + H] +, 751 [M + H + NBA] ⁺ ) confirms the expected structure.

Step 2: Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-arginine aldehyde

0.56 g (0.93 mmole) of tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy light yellow oil inin L-Arg-lactam (Example 2, first Step 2) is reduced as described in Example 1, Step 2, using proportional amounts of reagents and solvents, except that the subsequent chromatography is carried out in ethyl acetate [Rf (l) = 0.4 (lactome), 0.0 (aldehyde)]. 0.4 g (67%) of the title product is triturated with n-hexane. R / 6) = 0.4. Melting point: 91-93 ° C.

The FAB mass spectrum (600 [M + H] ⁺ , 753 [M + H + NBA] ⁺ ) confirms the expected structure.

Step 3: D-2-Cyclopentyl-2-hydroxyacetyl-L-prolyl-L-arginine aldehyde hemisulfate

0.38 g (0.63 mmole) of tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-prolyl-N ^G -benzyloxy-carbonyl-L-ar-Arginine aldehyde (Example 2, the second Step 1) was transformed as described in Example 1, Step 3 using proportional reagents and solvents to give the title product (0.22 g, 81%). HPLC (I): k '= 4.29 and 4.68.

The FAB mass spectrum (382 [M + H] +, 535 [M + H + NBA] ⁺ ) confirms the expected structure.

For example, the starting material is prepared as follows:

Preparation of Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-proline (THP-D-cPga-Pro)

Step A: Resolution of O-acetyl-DL-2-cyclopentyl-2-hydroxy-acetic acid with acylase I

0.72 g (5.0 mmol) of DL-2-cyclopentyl-2-hydroxy-acetic acid [M. Robba and Y. Le Guen, Chim. Therap. 1968, 238; Chem. Abst. 66, 18633f (1967)] in Example 1, A and

As described in Step B, acetylated or hydrolyzed with acylase I using proportional amounts of reagents and solvents to yield the resulting L-2-cycl

Separate the lopentyl 2-hydroxyacetic acid [R 1 (14) = 0.35] and O -acetyl D-2-cyclopentyl-2-hydroxyacetic acid [R 14 (0.52)], and the latter Example 1, Step C, was converted to D-2-cyclopentyl-2-hydroxy-acetic acid using proportional amounts of reagents and solvents. The products thus obtained:

a) 0.2 g (1.39 mmol) of L-2-cyclopentyl-2-hydroxyacetic acid, m.p. 108-109 ° C; [α] _D = -15.95 ° (c = 1 in methanol). Optical purity = 99% (chiral HPLC).

b) 0.16 g (1.1 mmol) of D-2-cyclopentyl-2-hydroxyacetic acid, m.p. 106 ° C;

[α] _D = + 15.5 ° (c = 1 in methanol). Optical purity = 98% (chiral HPLC).

Step B: D-2-Cyclopentyl-2-hydroxy-acetic acid (Resolution of DL with D-Tyrosine Hydrazide)

1.44 g (10 mmol) of DL-2-cyclopentyl-2-hydroxy-acetic acid [M. Robba and Y. Le Guen, Chim. Therap. 1968, 238; Chem. Abst. 66, 18633f (1967)] and 1.97 g (10 mmol) of D-tyrosine hydrazide are suspended in 50 ml of anhydrous ethanol and refluxed until completely dissolved. After the solution has cooled to room temperature, it is kept in the refrigerator overnight. The precipitated crystals were filtered off, washed with 2 x 2 mL of cold anhydrous ethanol and dried in a vacuum desiccator. The diastereomeric salt (2.14 g, 6.3 mmol) was recrystallized twice from 40 ml of anhydrous ethanol. The diastereomeric salt (1.54 g, 4.53 mmol) was dissolved in diethyl ether (10 mL) and 1M potassium bisulfate (5 mL). The diethyl ether layer was washed with water to anhydrous, dried over anhydrous sodium sulfate and then concentrated under a pressure of 2.0-2.5 kPa. The crystalline residue is triturated with petroleum ether, filtered and air-dried. 0.56 g (3.88 mmol, 76.7%) of D-2-cyclopentyl-2-hydroxyacetic acid is obtained. Melting point: 109-110 ° C. [α] _D = + 14.8 ° (c = 1 in methanol) and -30 ° (c = 1 in acetic acid). Optical purity = 98% (chiral HPLC). Analysis for C ₇ H ₁₂ O ₃ (144.17):

Calculated: C, 58.31; % H, 8.39;

Found: C, 58.56; H% = 8.50.

Step C: D-2-Cyclopentyl-2-hydroxy-acetic acid benzyl ester

0.47 g (3.2 mmol) of D-2-cyclopentyl-2-hydroxyacetic acid (Example 2, Step B) was converted to 0 using a proportion of reagents and solvents as described in Example 1, Step E, 74 g (95%) of the title compound are obtained in the form of an oil. Rf (12) = 0.20.

Step D: Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxy-acetic acid benzyl ester

D-2-Cyclopentyl-2-hydroxyacetic acid benzyl ester (0.74 g, 3.2 mmol) (Example 2, Step C) was prepared as described in Example 1, Step F, using proportional reagents and solvents. - we are transforming. The resulting evaporation residue [Rf (12) = 0.27-0.36] is considered to be 2.75 mmol of the title product.

Step E: Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxy-acetic acid

2.75 mmol of tetrahydropyranyl-D-2-cyclopentyl-2-hydroxy-acetic acid benzyl ester (Example 2, Step D) was converted as described in Example 1, Step G using proportional reagents and solvents.

Obtained as an oil (0.6 g, 2.6 mmol, 95%). Rf (l) = 0.25.

Step F: Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxy-acetyl-L-prolyl-benzyl ester

2.6 mmol of tetrahydropyranyl-D-2-cyclopentyl-2-hydroxy-acetic acid (Example 2, Step E) and 0.62 g (2.6 mmol) of L-proline benzyl ester hydrochloride were added.

Example 1 is condensed using proportional amounts of reagents and solvents as described in Step H. Obtained as an oil (0.97 g, 90%). R (l) = 0.7.

Step G: Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-proline

0.97 g (2.3 mmol) of tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-proline-benzyl ester (Example 1, Step F) was prepared as described in Example 1, Step I - Reaction with a proportion of reagents and solvents afforded 0.71 g (89%) of the title compound as an oil [Rj (9) = 0.5] which was used directly in Step 1 of the Example and converted to a crystalline salt. as follows.

Tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-proline (0.03 g, 0.1 mmol) was dissolved in diethyl ether (5 mL) and dicyclohexylamine (0.018 mL, 0.1 mmol) was added and the precipitated crystals were filtered off. , washed with diethyl ether and dried in a vacuum desiccator. 0.048 g (95%) of tetrahydropyranyl-D-2-cyclopentyl-2-hydroxyacetyl-L-proline-dicyclohexylammonium salt are obtained. Mp 145-147 ° C. Analysis calculated for C ₂₉ H ₅₀ N ₂ O ₅ (506.73): C, 68.73; % H, 9.95; % N, 5.52;

Found: C, 67.25; % H, 9.94; N, 6.20%.

The FAB mass spectrum (507 [M + H + DCHA] *) confirms the expected structure.

Methods

Ml. method

Plasma clot preparation

a) Plate-like plasma (PRP) is prepared by centrifuging a 9: 1 mixture of human blood and 3.8% sodium citrate water (1/2) at 240 x g for 5 minutes.

b) Add 200 μΐ of PRP to each reaction vessel, add 80 μΐ of 40 mM calcium chloride and allow to stand at room temperature for 1 hour. The resulting plasma clot was washed with 6 x 2 mL of 0.9% sodium chloride with gentle agitation and 5 minutes of sedimentation to remove enzymes remaining in solution (factor Xa and thrombin). The thrombin content of the wash was determined as follows.

c) To 400 μΐ wash solution, add 100 μΐ 1 mM Tos-GlyPro-Arg-pNA substrate solution, incubate for 30 min at 37 ° C and stop the reaction with 100 μΐ 50% acetic acid. 150-150 μί of the mixture was placed in wells of a 96-well microtitre plate and the extinction values were determined at 405 nm (ELISA READER SLT Laborinstrument GmbH, Austria). With effective washing, the extinction value is less than 5% of the control.

HU 222 199 Bl

M2. method

Determination of Plasma Clot Factor Xa Inhibition

a) 0.1-1.0-10 and 100 pg / ml solutions of peptide inhibitor are prepared in 0.1 M Tris buffer containing 0.02% human albumin pH 8.5.

b) After aspirating the wash fluid, 400 µl of peptide solution (3 replicate samples at each concentration) is added to the clots (Method M1) and 400 µl of buffer as a control and incubated for 5 minutes at 37 ° C. Subsequently, 100 µl of 2 mM Moc-D-Chg-Gly-Arg-pNA substrate solution was added, incubated for another 30 minutes at 37 ° C, and then quenched with 100 µl of 50% acetic acid.

c) 150-150 µL of each reaction mixture is placed in wells of a 96-well microtitre plate and the extinction values are determined at 405 nm (ELISA READER SLT Laborinstrument GmbH, Austria). From the mean extinction values relative to the control, the peptide concentration (IC ₅₀ ) required for 50% inhibition is plotted.

M3. method

Determination of inhibition of plasma clot thrombin

a) Prepare solutions of the peptide inhibitor according to M2. Method A (a).

b) After aspirating the wash fluid, 400 µl peptide solution (3 replicate samples at each concentration) is added to the clots (Method M1) and 400 µl buffer as a control and incubated for 5 minutes at 37 ° C. Subsequently, 100 µl of 1 mM Tos-Gly-Pro-Arg-pNA substrate solution was added, incubated for another 30 minutes at 37 ° C, and then quenched with 100 µl of 50% acetic acid. Hereinafter referred to as M2. (c).

M4. Method Fibringel

a) Add 200 µl of 6 mg / ml human fibrinogen (SIGMA), 25 µl of 25 NIH U / ml human thrombin (SIGMA) and 40 µl of 100 mM calcium chloride to each reaction vessel and allow to stand for 1 h. for 20 hours at 20-22 ° C. The resulting fibrin gel was washed with 3 x 2 ml of physiological saline solution with gentle stirring and 5 minutes of sedimentation to remove any remaining thrombin in the solution. Washing performance is determined by Ml. (c).

M5. method

Determination of inhibition of fibrin gel bound thrombin

a) Peptide inhibitors were prepared in 0.1-1.0-10 and 100 pg / ml solution in Hepes / NaCl buffer (0.01 M Hepes and 0.1 M sodium chloride, pH 7.4).

(b) hereinafter referred to as "M3",. (b).

M6. method

Measurement of Factor Xa Inhibition in 96-well Microtitre Plate

a) Factor Xa (human, SIGMA) solutions were prepared at 1.3 U / ml and peptide inhibitors at 0.1-1.0-10 and 100 pg / ml in phosphate buffer (0.1 M sodium phosphate and 0.05 ml). M sodium chloride, pH 7.4). A 0.33 nM distilled aqueous solution of the BzIle-Glu-Gly-Arg-pNA substrate was used.

b) 33 reaction mixtures were prepared from the control and each peptide solution. 30 µl of peptide was added to the wells of the plate as well as buffer, 30 µl of Factor Xa, 90 µl of buffer and 150 µl of substrate, and after 10 minutes the extinction values were read at 405 nm. Hereinafter referred to as the M2 method

(c).

Claims (8)

PATIENT INDIVIDUAL POINTS 1. New peptidaldehyde derivatives of formula (I) D-Xaa-Pro-Arg-H (I) wherein Xaa is 2-cycloheptyl-2-hydroxyacetyl or 2-cyclopentyl-2-hydroxyacetyl; Pro is L-proline radical and Arg is L-arginine radical and its acid addition salts with organic or inorganic acids. 2. D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-Larginine aldehyde, and acid addition salts thereof. 3. D-2-cycloheptyl-2-hydroxyacetyl-L-prolyl-Larginine aldehyde hydrogen sulfate. 4. D-2-cyclopentyl-2-hydroxyacetyl-L-prolyl-Larginine aldehyde and acid addition salts thereof. 5. D-2-cyclopentyl-2-hydroxyacetyl-L-prolyl-Larginine aldehyde hydrogen sulfate. 6. A pharmaceutical composition comprising as active ingredient a compound of formula (I), wherein Xaa, Pro and Arg are as defined in claim 1, or a pharmaceutically acceptable salt thereof, a solvent, diluent, carrier commonly used in medicine. and with fillers. Compounds of formula I wherein Xaa, Pro and Arg are as defined in claim 1 for use as a medicament. Use of compounds of formula I wherein Xaa, Pro and Arg are as defined in claim 1 for the preparation of an anticoagulant.